JP3393220B2 - Fluorescent probe for active oxygen detection - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention is excited by visible light, which is useful as a probe for detecting changes in the oxidative state of active oxygen and the like, particularly changes in the oxidative state of cells and tissues , and
Fluorescent oxidative probe that emits in water, especially hydroxyradi
The present invention relates to a fluorescent oxidative probe having organic phosphorus that reacts sensitively with active oxygen such as cal .

[0002]

2. Description of the Related Art Conventionally, fluorescent or chemiluminescent reagents have been developed for the purpose of chemically detecting active oxygen. It is said that the detection method using these reagents has a high sensitivity, is very effective as a method for detecting a short-lived active oxygen which is produced in a small amount in a living body. However, there are problems with the specific detection of specific reactive oxygen species. In other words, the reaction of active oxygen is diverse, and each active oxygen has unique reaction characteristics and substrate specificity, and at the same time, it often shows common reactivity and specifically detects individual active oxygen. Since the design of the reagent is very difficult, it is necessary to use the detection reagent together with an active oxygen inhibitor, for example, an enzyme such as superoxide dismutase (SOD) or catalase, in order to identify the reactive species. It is said. However, since the enzyme cannot pass through the cell membrane, it is difficult to identify the active oxygen produced in the cell. On the other hand, using a detection reagent specific to active oxygen, a method for directly detecting a species of active oxygen, which does not require an auxiliary reagent such as the enzyme, has been proposed, and is useful for such a method. Development of detection reagents has been carried out. The following compound 1 has been proposed as such a reagent.

[0003]

[Chemical 1]

The generation of active oxygen can be considered to be a local and time-dependent change in the oxidative state in cells or aggregates thereof in a living body, and the dynamics of the active oxygen in a living body is elucidated. For this purpose, it is necessary to detect active oxygen in cells and tissues in a living state. As such a method, an imaging method has been proposed, and a reagent capable of observing a physiological phenomenon of a living body more faithfully and without causing damage to the living body has been developed.

As such a reagent, dichlorodihydrofluorescein (H2DCFDA = dichlorodihydrofluorescein) of the following two compounds, which are dihydro forms of fluorescein and rhodamine, is used.
Dihydrorhodamine (dihydororhodamine123) has been proposed.

[0006]

[Chemical 2]

The compounds exemplified above are completely colorless and non-fluorescent compounds, but when oxidized by active oxygen, they are converted into fluorescein and rhodamine which have strong fluorescence.
Moreover, since they can be excited by long wavelengths, they are less likely to damage the living body, and they are not affected by autofluorescence of coexisting proteins. Therefore, they can be said to be suitable for the imaging method. Further, the esterified derivative passes through the cell membrane and is converted into the original compound by intracellular esterase. When the ester is removed, it is highly water-soluble and cannot pass through the cell membrane, so that the dye can remain in the cell and the production of active oxygen localized in the cell can be examined. In the case of rhodamine, which has a positive charge, it is known that it accumulates in membrane components and mitochondria in cells, and using this property, imaging of active oxygen generated in mitochondria was performed. There is. However, it has a drawback that it is not a reagent specific to a specific reactive oxygen species.

As mentioned above, various reagents have been developed. However, some examples have realized specificity of reaction with active oxygen and long-wavelength and strong fluorescence / luminescence required for imaging. Almost never exist. Thus, active oxygen, in particular O ₂ ^- H due to (superoxide) generation
_If it becomes possible to selectively detect ₂ O ₂ , .OH or a peroxide of a biological component, preferably in a living state, it is useful for knowing the influence on the living body. By the way, the above-mentioned reagent has a portion in the molecule that is oxidized by active oxygen (hereinafter sometimes referred to as “reaction portion”).
And a portion (hereinafter, sometimes referred to as a “fluorophore”) whose absorption of light and fluorescence characteristics change with the above-mentioned oxidation, so that the peroxide species can be specifically detected. Must have substrate specificity in the reactive moiety.

[0009]

Therefore, the object of the present invention is to enable the specific detection of peroxide species,
It is an object of the present invention to provide a fluorescent probe that can be excited by visible light and that can be observed by imaging. By the way, it is known that triphenylphosphine or a compound in which one benzene ring is changed to pyrene efficiently reacts with a peroxide such as lipid peroxide.
At that time, since optical characteristics such as absorption characteristics change, there is a method of using the characteristics as a reagent for detecting lipid peroxide, and an example used for HPLC analysis by absorption method has been published (U. Pinkernell, S. .Effkemann, U.Karst: Anal.Ch
em., 69, 3623-3627 (1997)). The latter compound is used as a reagent utilizing the property of producing a fluorescent substance by being oxidized. Does not have hydrophilicity 2. 2. The excitation wavelength of the fluorescent substance is a short wavelength, and 3. It is insufficient as a fluorescent probe because it has no specificity for peroxide species. Therefore, the present inventor has found that the reaction of the phosphorus atom with the peroxide and the excitation with visible light,
We thought that a fluorescent probe with improved specificity could be obtained by combining it with a fluorescent compound that emits light in 1 ), and synthesized various compounds and examined their properties.

[0010]

The gist of the present invention is to excite one phenyl group of triphenylphosphine with visible light.
And Fluorescein-like compound residue that emits light in water
Is a fluorescent probe for detecting active oxygen , preferably the aforementioned fluorescein-like compound is an unsubstituted hydroxyphenylfluorone, which is a fluorescent probe for detecting active oxygen. The present inventor has aromatic phosphines of the active oxygen reaction sites, variable
The above problem is solved by combining with a fluorescent probe that is excited by visible light and emits light in water .

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail. Synthesis of Compounds of TPP-HFs and TPPO-HFs of the Present Invention As specific compounds, those represented by the following structural formula 3 can be mentioned.

[0012]

[Chemical 3]

A: TTP-HF1 and TPPO-HF
Synthesis of 1. Synthesis of 2,2 ', 4,4'-tetrahydroxybenzophenone 1. β-resorcinic acid 6.00 g (38.9 mmol), resorcinol 6.10 g (55.4 mmol), freshly dried zinc chloride 25 g
(180 mmol) and phosphorus oxychloride 32 ml (340 mmol) were stirred at room temperature for 12 hours, the reaction solution was poured into ice water, and the precipitate was collected by filtration.
2 ', 4,4'-Tetrahydroxybenzophenone was obtained. Yield, 8.40 g, yield 87.3%, recrystallized from water, red needle crystals, melting point, 196-198 ° C. ¹ H-NMR (300 MHz, DMSO-d ₆ ) δ: 6.30 (2H, d, J = 2.2H
z) 6.31 (2H, dd, J = 2.2,9.2Hz), 7.71 (2H, d, J = 9.2Hz), 11.
26 (4H, br) 2. Synthesis of 3,6-dihydroxyxanthe-9-one 2,2 ', 4,4'-tetrahydroxybenzophenone 1.00 g (4.03
(50 mmol) and add 50 ml of water to the autoclave for 2 days.
Heat to 20 ° C. After allowing to cool to room temperature, the generated precipitate was collected by filtration to obtain 3,6-dihydroxyxanthe-9-one. Orange powder, yield 720 mg, yield 76.9% ¹ H-NMR (300 MHz, DMSO-d ₆ ) δ: 6.82 (2H, d, J = 2.0H
z) 6.86 (2H, dd, J = 2.2,8.6Hz), 7.98 (2H, d, J = 8.6Hz), 10.
75 (2H, s) 3. Synthesis of 3.6-bis (t-butyldimethylsilyloxy) xanthe-9-one 1.73 g (7.50 mmol) 3,6-dihydroxyxanthe-9-one are dissolved in anhydrous dimethylformamide. Imidazole 5.10g
(75.0 mmol), t-butyldimethylchlorosiloxane 6.75
g (45.0 mmol) is added and stirred at room temperature for 2 hours. The reaction solution was diluted with toluene, washed sufficiently with water, added with MgSO ₄ and dried, and evaporated under reduced pressure in vacuo to obtain 3.6-bis (t-butyldimethylsilyloxy) xanthe-9-one as a white solid. Yield 3.35 g, yield 96.5%, recrystallized from ethanol, colorless needle crystals, melting point 151-154 ° C. ¹ H-NMR (300 MHz, CDCl ₃ ) δ: 0.29 (s, 12H) 1.01.
(S, 18H), 6.83-6.87 (m, 4H), 8.20 (d, 2H, J = 7.3Hz) 4. Synthesis of TPP-HF1 4-bromophenyldiphenylphosphine 102 mg
(0.30 mmol) is dissolved in 6 ml of anhydrous 2-methyltetrahydrofuran. It is immersed in a liquid mixture of liquid nitrogen and isopentane and cooled to -150 ° C. 0.22 ml of t-butyllithium (1.5 M, n-pentane solution)
(0.33 mmol) is added to lithiate the halogen. To this, a solution prepared by dissolving 274 mg of 3.6-bis (t-butyldimethylsilyloxy) xanthe-9-one synthesized in 1.3) in tetrahydrofuran was added dropwise, and the temperature was kept at -130 ° C for 30 minutes. Stir. After that, water is added to the reaction solution to decompose the lithium salt, and the temperature is lowered to room temperature. The organic layer was extracted twice with ethyl acetate, washed with saturated brine, dried over Na ₂ SO ₄ , and the solvent was distilled off under reduced pressure. Acetic acid was added to the residue, and the mixture was stirred at room temperature to cleave the protective group and dehydrated to give TPP as a product.
-HF1 was obtained. Acetic acid is removed by distillation under reduced pressure in a vacuum, and a silica gel column (dichloromethane: methanol) is used. Yield 59 mg, yield 41.7%, recrystallized from dichloromethane: methanol, orange needle crystals ¹ H-NMR (300 MHz, DMSO-d ₆ ) δ: 6.63 (m, 4H), 7.06
(D, 2H, J = 9.3Hz), 7.3-7.5 (m, 14H), FAB MASS 473 (M ⁺ ＋
1) 5. Synthesis of TPPO-TF1 47 mg (0.1 mmol) of TPP-HF1 is dissolved in methanol, 30% H2O2 aqueous solution is added, and the mixture is stirred at room temperature. H ₂ O ₂ and methanol were removed by vacuum distillation to remove TP
PO-HF1 is obtained quantitatively. Recrystallized from ethanol,
Red needle crystal ¹ H-NMR (300 MHz, DMSO-d ₆ ) δ: 6.47 (d, 2H, J = 2.0H
z), 6.50 (dd, 2H, J = 2.1,9.2Hz), 6.97 (d, 2H, L = 9.2), 7.5-
7.9 (m, 14H), FAB MASS 489 (M ⁺ +1) TPP-HF and TPPO-HF
And 5 are shown.

[0014]

[Chemical 4]

[0015]

[Chemical 5]

B: TPP-HF2 and TPPO-HF
Synthesis of 2 Synthesis of 5-chloro-β-resorcinic acid 25 g (0.16 mol) of β-resorcinic acid was added to 40 parts of acetic acid.
Dissolve in 0 ml, 13 ml of SO ₂ Cl ₂ (0.16 mo
l) is added and stirred at room temperature. After completion of the reaction, water was added to the reaction solution and extracted three times with ethyl acetate, the ethyl acetate layer was washed with saturated brine and dried over Na ₂ SO ₄ , and the solvent was evaporated under reduced pressure to obtain the desired product. Was obtained. Yield 22 g, yield 72.3%, recrystallized from water, colorless needle crystals ¹ H-NMR (300 MHz, DMSO-d ₆ ) δ: 6.47 (1H, s), 7.66
(1H, s), 11.20 (2H, s), 11.22 (1H, br) 2. Synthesis of 5,5'-dichloro-2,2 ', 4,4'-tetrahydroxybenzophenone 5-chloro-β-resorcinic acid 19g (98mmol), 4-chlororesorcinol 23g (160mmol), freshly dried zinc chloride Five
6 g (410 mmol) and 93 ml of phosphorus oxychloride (990 mmol) are heated to 70-80 ° C for 1 hour. The reaction solution was poured into ice water, and the precipitate was collected by filtration to obtain 5,5'-dichloro-2,2 ', 4,4'-tetrahydroxybenzophenone. Yield 5.1 g, yield 16.5%,
Recrystallized from water, red needle crystal ¹ H-NMR (300 MHz, DMSO-d ₆ ) δ: 6.55 (2H, s) 7.28
(2H, s), 11.16 (2H, s), 11.22 (2H, s) 3.Synthesis of 2,7-dichloro-3,6-dihydroxyxanthe-9-one 5,5'-dichloro-2,2 50 ml of water was added to 1.3 g (4.1 mmol) of ', 4,4'-tetrahydroxybenzophenone and 2,7-dichloro-3,6-as in the case of 3,6-dihydroxyxanthe-9-one.
Dihydroxyxanthe-9-one was obtained. Yield 1.0 g, yield 84.2% ¹ H-NMR (300 MHz, DMSO-d ₆ ) δ: 7.03 (2H, s) 8.00
(2H, s), 11.21 (2H, s) 4. 3.6-bis (t-butyldimethylsilyloxy) -2,7-
Synthesis of dichloroxanthe-9-one 2,7-dichloro-3,6-dihydroxyxanthe-9-one 777mg
(2.62 mmol) is dissolved in 70 ml of anhydrous dimethylformamide.
Imidazole (1.78 g, 26.2 mmol) and t-butyldimethylchlorosiloxane (2.37 g, 15.7 mmol) were added, and 3.6-bis (t-t) was prepared in the same manner as 3.6-bis (t-butyldimethylsilyloxy) xanthe-9-one. Butyl dimethyl silyloxy)
-2,7-Dichloroxanthe-9-one was obtained. Yield 1.26
g, yield 91.6%, recrystallized from ethanol, colorless needle crystal ¹ H-NMR (300 MHz, CDCl ₃ ) δ: 0.33 (s, 12H) 1.07
(S, 18H), 6.90 (s, 2H), 8.29 (s, 2H) 5. Synthesis of TPP-HF2 4-Bromophenyldiphenylphosphine 307 mg
(0.90 mmol) is dissolved in 18 ml of anhydrous 2-methyltetrahydrofuran. t-butyllithium (1.5
M, n-pentane solution) 0.66 ml (0.99 mmo
l), 3.6-bis (t-butyldimethylsilyloxy) -2,7
-Dichloroxanthe-9-one 822 mg (1.8 mmo
1) using a tetrahydrofuran solution of TPP-HF1
TPP-HF2 was obtained in the same manner as. Yield 40.3m
g, yield 8.3%, recrystallized from dichloromethane: methanol, red needle crystals ¹ H-NMR (300 MHz, DMSO-d ₆ ) δ: 6.48 (s, 2H) 7.00
(S, 2H), 7.3-7.5 (m, 14H), FAB MASS 542 (M ⁺ +1) 6. Synthesis of TPPO-HF2 54 mg (0.1 mmol) of TPP-HF2 is dissolved in methanol, 30% H2O2 aqueous solution is added, and the mixture is stirred at room temperature. H ₂ O ₂ and methanol were removed by distillation under reduced pressure to remove T
PPO-HF2 is obtained quantitatively. Recrystallization from dichloromethane: methanol, dark red needle crystal ¹ H-NMR (300 MHz, DMSO-d ₆ ) δ: 6.62 (s, 2H) 7.04
(S, 2H), 7.6-7.9 (m, 14H), 10.79 (s, 1H), FAB MASS 558 (M
⁺ +1)

Physical Properties of TPP-HFs A: Fluorescence Spectra of Synthesized TPP-HFs and TPPO-HFs Three-dimensional fluorescence spectra of the synthesized TPP-HFs and TPPO-HFs were measured. The measurement was carried out in 0.1 M sodium phosphate buffer (pH 7.8) using TPP-HF,
It was performed at a concentration of 10 μM of TPPO-HFs. The result is shown in Figure 1.
(I) and (II) of FIG. 2 and (I) and (II) of FIG. TP
In both cases of P-HF1 and TPP-HF2, the TPPO-HFs, which are the oxidants, had significantly higher fluorescence intensity under neutral conditions.

PH dependence of fluorescence of TPP-HFs and TPPO-HFs In order to use TPP-HFs under physiological conditions, it is necessary to show stable fluorescence in a neutral region.
The pH dependence of fluorescence of TPPO-HFs was examined. The measurement was performed at 20 ° C. in a 50 mM sodium phosphate solution. 50 mM Na ₃ PO ₄ .12H ₂ O, Na ₂ HPO ₄
12H ₂ O, NaH ₂ PO ₄ , 12H ₂ O, H ₃ PO ₄ aqueous solutions were prepared and mixed at arbitrary ratios to prepare phosphoric acid solutions having various pH values. 2mD of each compound immediately before measurement
MF solution was added and fluorescence was measured. TPP-HF,
The final concentration of TPP0-HFs was 1 μM. The measurement results are shown in FIGS. 3 and 4. Excitation wavelength and fluorescence wavelength are TPP-HFl (Ex.495mm / Em.520nm) and TP, respectively.
PO-HFl (Ex.495nm / Em.525nm), TPP-HF
2 (Ex. 507 nm / Em. 527 nm), TPPO-HF2 (Ex.
5O9nm / Em. 533 nm).

In Kaleida Graph, match the following formula (f
It) and the pKa value was calculated. The results are shown in Table 1. Calculation of pKa value F = Fmax / (1 + 10 ^(pKa-pH) ) + Fmin / (1
+10 ^(ph-pKa) ) F = Fluorescence Intensity Fmax = Maximum Fluorescence Inten
sity) Fmin = Minimum Fluorescence Inten
sity)

[0020]

[Table 1]

From the above-mentioned FIGS. 3 and 4, TPP-HFs have remarkably small fluorescence intensity in the acidic region as compared with pH 8.
On the contrary, it was found that the alkaline region had a fluorescence intensity similar to that of TPPO-HFs. As mentioned above, pH
The difference in fluorescence intensity observed in the phosphate buffer of 7.4 is a phenomenon depending on the pKa value of each compound of TPP-HF and TPPO-HF, and as a result, TPPO
It was revealed that the fluorescence intensity increased near neutral by changing to -HF.

Concentration dependence of fluorescence of TPPO-HFs Possibility of quantitative measurement Whether TPP-HFs can be used for the purpose of quantification, TP
It was examined by examining the concentration dependence of the fluorescence intensity of PO-HFs. The fluorescence intensity was measured by changing the concentration of TPPO-HFs in a sodium phosphate buffer of pH 7.4. Results are shown in FIG. It was revealed that the fluorescence of TPPO-HFs correlates with the concentration of TPPO-HFs and can be used for quantification.

As a result of the above studies, it has been confirmed that the excitation / fluorescence wavelengths are long and that TPPO-H in the vicinity of neutral is
Since the fluorescence intensity of Fs is stable, TPP-H
TPP-HF2 is superior to F1 as a fluorescence detection reagent.

Reaction of TPP-HF1 and active oxygen (xanthine / xanthine oxidase system) 1) Xanthine / xanthine oxidase (Xanthine / Xanthine Oxida)
se) system. Xanthine oxidase (XO) has a molecular weight of 275,00
Consists of 0 and 2 units, 1 in 1 unit
It has one Mo ion, one FAD, and two non-heme iron-sulfur proteins. X. O. Simultaneously oxidizes xanthine (X) or hypoxanthine to uric acid, and simultaneously produces superoxide (O ₂ ⁻ ) and hydrogen peroxide (H ₂ O ₂ ) from oxygen. The following 6 is the chemical reaction.

[0025]

[Chemical 6]

This system is best known as an active oxygen producing system and is a typical oxidase producing active oxygen. However, the details of the generation mechanism of active oxygen are unclear, and it is not well known that H ₂ O ₂ is directly generated from the (XO + X) system.
On the other hand, when H ₂ O ₂ and superoxide (O ₂ ⁻ ) are present at the same time, a small amount of iron ion catalyst causes Ha
The Ber-Weiss reaction proceeds to generate a hydroxyl radical (.OH). The reaction is represented by reaction formula A. Scheme ^{_{A H 2 O 2 + Fe 2+}} - → · OH + OH- + Fe 3+ O 2+ Fe 3+ ---- → O 2 + Fe 2+ i.e., the xanthine / xanthine oxidase system H ₂ O ₂ and O ₂ ^- In addition, .OH is also produced by the iron ion catalyst contained in the salt of the buffer in a trace amount.

As described above, in the xanthine / xanthine oxidase system, H ₂ which is the main active oxygen produced in the living body
^{_{O 2, O 2 -, ·}} OH is present at the same time, T in this system
By applying PP-HF1, it was possible to investigate the reactivity with active oxygen under physiological conditions, and it was decided to investigate. At the same time, by adding catalase and superoxide dismutase (SOD), which are inhibitors of H ₂ O ₂ and O ₂ ⁻ , respectively, it is also possible to determine how TPO-HF1 reacts with specific reactive oxygen species. I examined it. Catalase, H by SDO ₂ O _2, O ₂ ^-
The mechanism by which each is inhibited is as shown in the reaction formula of B below. X. O. The ratio of O ₂ ⁻ and H ₂ O ₂ produced from the above varies greatly depending on the conditions. For example, as the enzyme concentration increases, O
The production ratio of ₂ ⁻ increases and H ₂ O ₂ decreases. It also depends on the substrate concentration. It is also affected by pH. Here, the experiment was performed according to the conditions shown by Fridovich et al. Under this condition, O ₂ ⁻ and H ₂ O ₂ are produced in equal amounts (about 1.2 μM / min.).

2) Examination of reactivity of TPP-HF1 TPP-HF1 was applied to a xanthine / xanthine oxidase system under the conditions shown by Fridovich et al., And reactivity with active oxygen was examined. Buffer containing xanthine 50 μM (0.1 mM EDTA
50 mM potassium phosphate buffer, pH 7.
In 8), a solution was prepared so that the final concentration of TPP-HF1 was 10 μM. O. Was added to start the reaction.
FIG. 6 shows the result of measuring the time change of the fluorescence intensity of TPPO-HF1 produced by oxidation. As seen in a), X. O. The addition increased the fluorescence intensity over time. b) X. O. No change in fluorescence intensity was observed when no was added (blank). _{Further, c) H 2 0 2,} O 2 - Catalase is the inhibitor, d) the addition of SOD, increase in fluorescence intensity was suppressed. As a control experiment, the addition of the enzyme deactivated by heating did not affect the fluorescence intensity.

Since the oxidation was suppressed by catalase, it can be seen that H ₂ O ₂ is necessary for the reaction. On the other hand, SOD carries out a disproportionation reaction of O ₂ ⁻ to produce H ₂ O ₂ . If H ₂ O ₂ causes oxidation, SOD
Is expected to accelerate the oxidation reaction. However, since the increase in fluorescence intensity was suppressed by SOD as a result of d), it is suggested that O ₂ ⁻ is also required for the oxidation reaction. The amount of active oxygen generated in the xanthine / xanthine oxidase system is as described above.
The concentration is as low as several μM per minute, and under such conditions, the oxidation reaction of TPP-HF1 hardly progresses with H ₂ O ₂ or O ₂ ⁻ , and the observed increase in the fluorescence intensity of TPPO-HF1. Is likely to be mainly due to OH.
In addition, X. O. The fact that the fluorescence starts to increase after a short period of time after the addition of S and the change in the fluorescence intensity draws an S-shaped curve also suggests that the reaction is due to the secondarily generated .OH. From the above, TPP-HF acids are ^{_{H 2 O 2, O 2 -}} ,
Among the three types of active oxygen, OH, it has a high reactivity with OH, and it has been revealed that it has specificity with OH. Although a protein coexists as an enzyme in this system, the fluorescence of TPP-HF1 was not affected by the protein.

3) Confirmation of TPPO-HF1 production TPPO by xanthine / xanthine oxidase system
The process of -HF1 production was followed by changes in the fluorescence spectrum. Similar to the condition of 2) above, X. O. The reaction was started by addition of. The result of measuring the fluorescence spectrum every few minutes after the start is shown in FIG. As the oxidation reaction progresses, the fluorescence intensity increases and the maximum fluorescence wavelength becomes 5
nm shifts to longer wavelength side, and finally TPPO-HF
It was in agreement with the spectrum of l. This result is the above TPP
-In agreement with the physical properties of HF and TPPO-HF,
As a result of the reaction, it was confirmed that TPPO-HF1 was produced.

Reaction of TPP-HF1 with various active oxygen species (chemical reaction system) Reaction of TPP-HF1 with hydrogen peroxide In the examination of the above-mentioned xanthine / xanthine oxidase system, TPP-HF has a high reaction with a hydroxyl radical. Although it showed good properties and hardly reacted with hydrogen peroxide, hydrogen peroxide was directly added here to examine the reactivity with TPP-HF1. TPP-HF1 was dissolved in 50 mM potassium phosphate buffer (pH 7.8) so that the final concentration was 10 μM, and changes with time of fluorescence at an excitation wavelength of 495 nm and a fluorescence wavelength of 525 nm were measured. After 3 minutes, hydrogen peroxide was added to start the reaction. The change in fluorescence with time after the start of the reaction is 60
Measured for minutes. The results are shown in Fig. 8. From the results, TPP-
It was found that HF1 has a very slow reaction rate with respect to 10 μM hydrogen peroxide. In the case of producing only a few μM of hydrogen peroxide like the xanthine / xanthine oxidase system, almost no increase in fluorescence is observed, and in consideration of application in vivo, what is present is μM order hydrogen peroxide. Hardly reacts.

Reaction of TPP-HF1 with hydroxyl radical 1) Fenton reaction As a system for producing a hydroxyl radical in a chemical reaction system, a Fenton reaction H ₂ O ₂ + Fe ²⁺ ---- → OH + OH ⁻ + Fe ³⁺ (F
Enton reaction) is the most famous. The difference from the Haber-Weiss reaction is that the reducing agent, superoxide, does not exist, and the divalent iron ion (Fe ²⁺ ) reacts with hydrogen peroxide to react with the trivalent iron ion (Fe ³⁺ ) And a hydroxyl radical are generated. Although Fe ²⁺ is relatively stable in pure water, it is extremely unstable in a neutral phosphate buffer, and is rapidly oxidized by oxygen within 30 seconds (autooxidation). Therefore, the chelating agent DTPA (diethylenetriamine) is used to protect Fe ²⁺ from autoxidation.
pentaacetate) was used.

2) TPP-HF reactivity was measured by preparing a solution of TPP-HF1 in 50 mM potassium phosphate buffer containing 0.1 mM to a final concentration of 10 μM, and Fe ²⁺ and peroxidation. Hydrogen was added simultaneously to start the reaction. Fe ²⁺ was 50 μM and the hydrogen peroxide concentration was 10 μM. Fe ²⁺ is highly pure Fe (N
H ₄ ) ₂ (SO ₄ ) ₂ was used. A blank to which only Fe ²⁺ or hydrogen peroxide was added was also measured. The results are shown in Fig. 9. (A in the figure) shows the case of simultaneous addition of Fe ²⁺ and hydrogen peroxide, b) shows the case of only hydrogen peroxide,
c) is Fe ²⁺ only and d) is blank. ) In the system a), the fluorescence intensity rapidly increased by adding Fe ²⁺ and hydrogen peroxide and reached the maximum value. The difference is clear when compared to the case of b) with hydrogen peroxide alone and the case of c) with only Fe ²⁺ added.

From the results of the Fenton reaction, TPP-H
F1 has a high reactivity to hydroxyls radicals, very small amount of active oxygen generated in vivo _{(H 2 O 2, O 2} -, ·
It was shown that OH) specifically reacts with a hydroxyl radical.

Evaluation of Reactivity of TPP-HF1 with Reactive Oxygen Species As described above with respect to the reactivity of TPP-HF1 with respect to each active oxygen species, TPP-HF1 has a high reactivity with .OH. It was confirmed qualitatively. In order to quantitatively evaluate the selectivity for reactive oxygen species, active oxygen 10
The amount of increase in the fluorescence intensity of TPP-HF1 per μM was compared. In the Fenton reaction, the change in fluorescence intensity at the stage when the reaction with .OH generated from 10 μM hydrogen peroxide was completed was defined as 1, and the change in fluorescence intensity per 10 μM of each reactive oxygen species was shown. Since O ₂ ⁻ has a life of about 5 seconds, it was determined from the amount of increase in fluorescence intensity immediately after addition. Singlet oxygen ( ¹ O ₂ ) and NO are endoperoxide E, respectively.
P-1 (endoperoxide) as NO generator NOC1 ₃
((Hrabie, JA, J.Org. Chem., 58, pp.1472-1476, 1
993) was used. From each half-life (25 minutes and 13.7 minutes), estimate 10 μM active oxygen and calculate TP
Relative ratio of reactivity to P-HF1, ie, · OH
The increase in the fluorescence intensity of TPP-HF1 was determined as 1, and the increase in the fluorescence intensity due to other active oxygen was determined as a relative ratio R. The values are shown in Table 2 below.

[0036]

[Table 2]

[0037]

Industrial Applicability As described above, it was confirmed that TPP-HFs react very sensitively with a hydroxyl radical, and thus a fluorescent oxidation probe specific to a specific reactive oxygen species can be designed. It is understood that by designing the combination of the structure of the reaction part and the fluorescent part (including selection of substituents), it is possible to design a fluorescent oxidation probe applicable to detection of a specific reactive oxygen species at a specific generation site. It is useful in that it has paved the way.

[Brief description of drawings]

FIG. 1 (I) is a three-dimensional fluorescence spectrum of TPP-HF1, and (II) is a three-dimensional fluorescence spectrum of TPPO-HF1.

FIG. 2 (I) is a three-dimensional fluorescence spectrum of TPP-HF2, and (II) is a three-dimensional fluorescence spectrum of TPPO-HF2.

FIG. 3: pH dependence of fluorescence of TPP-HF1 and TPPO-HF1

FIG. 4 pH dependence of fluorescence of TPP-HF2 and TPPO-HF2

FIG. 5: Concentration dependence of fluorescence intensity of TPPO-HFs

FIG. O. Change in fluorescence intensity due to TPPO-HF1 formation after addition of

FIG. 7: X. O. Fluorescence spectrum every few minutes after the start of the reaction by addition of

FIG. 8: Time-dependent change in fluorescence after initiation of reaction by addition of hydrogen peroxide

FIG. 9: Reactivity of hydroxyl radical with TPP-HF1

Claims (2)

(57) [Claims] 1. One phenyl of triphenylphosphine.
The fluorescein group is excited by visible light and emits light in water.
Active oxygen composed of compounds converted into sane analogs
Fluorescent probe for detection. 2. A claims fluorescein analogs is characterized in that it is a hydroxyphenyl phenylfluorone unsubstituted
Item 2. A fluorescent probe for detecting active oxygen according to item 1 .